The current model states that immunological memory in the CD4 T-cell compartment is mediated by an expanded population of antigen-specific T-cells that stably express a prototypic set of surface molecules and a potential to produce lymphokines that facilitate antigen elimination such as IFN-gamma and IL-5. However, this model may not accurately describe the in vivo situation because recent studies have shown that phenotypic and functional changes may not be stable features of antigen-experienced CD4 T-cells in the body. In addition, much of what is currently know about the behavior of memory T-cells is based on in vitro cultures that do not reproduce the complex in vivo environment in which secondary responses occur. Thus, the critically important mechanisms responsible for immunological memory must be studied using systems in which antigen-specific CD4 T-cells can be identified throughout the body at precise times after antigen administration based solely on expression of the appropriate T-cell receptor (TCR). Here they will use such a system to test the hypothesis that stimulation by residual antigen from the first exposure, causes antigen-experience CD4 T-cells to express the activated-surface phenotype and lymphokine profile, and to recirculate through non-lymphoid tissues. It is proposed that secondary responses are more efficient during this period because antigen-experienced CD4 T-cells are capable of IFN-gamma and IL-4 production in the non-lymphoid tissue where antigen enters into the body. In contrast, it is proposed that after residual antigen disappears, the antigen-experienced CD4 T-cells revert to the naive-surface phenotype and lymphokine profile, and recirculate through lymphoid tissues. It is proposed that secondary immune responses are more efficient during this period because antigen-specific antibodies increase the pace of antigen-presentation by targeting antigen to antigen-presenting cells in the lymphoid tissues for stimulation of the numerous, but essentially naive, antigen-experience T-cells. These hypotheses will be tested in two specific aims with new technologies that they have developed in the last project period. Immunohistochemical staining of whole mouse sections with an anti-clonotypic antibody will be used to define the distribution of antigen-experienced CD4 T-cells in the entire body at various times after the first or second exposure to antigen. Two -and three-color immunohistochemical staining with antibodies specific for antigen-specific CD4 T-cells, antigen-presenting cells, and lymphokines will be used to identify the sites in the body where antigen is presented to antigen-experienced T-cells when administered at various times after the first exposure, where and which lymphokines are produced by the T-cells, and how the presence of antigen-specific antibodies affects these events. Whole body, in situ detection of antigen-experienced CD4 T-cells has the potential to produce the first picture of the secondary immune response as it occurs in all parts of the body.